Dip1 Defines a Class of Arp2/3 Complex Activators that Function without Preformed Actin Filaments  Andrew R. Wagner, Qing Luan, Su-Ling Liu, Brad J. Nolen  Current Biology  Volume 23, Issue 20, Pages 1990-1998 (October 2013) DOI: 10.1016/j.cub.2013.08.029 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Dip1 Is a Potent Activator of Arp2/3 Complex (A) Domain organization of human SPIN90, budding yeast Ldb17, and fission yeast Dip1. The leucine-rich domain (LRD) is conserved in all species. The LRD does not have sequence homology to leucine-rich repeat domains. (B) Time course of polymerization of 3 μM 15% pyrene actin with or without 50 nM S. pombe Arp2/3 complex and a range of concentrations of Dip1. (C) Plot of maximum polymerization rate versus Dip1 or Wsp1-VCA concentration for pyrene actin polymerization assays described in (B). (D) Plot of calculated number of barbed ends versus time for reactions described in (B). Micromolar concentration of Dip1 in each reaction is indicated. (E) Pyrene actin polymerization assays with Arp2/3 complex and pyrene actin as in (B), plus 1 μM Dip1 and 200 nM Wsp1-VCA as indicated. See also Figure S1. Current Biology 2013 23, 1990-1998DOI: (10.1016/j.cub.2013.08.029) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Dip1-Mediated Activation of Arp2/3 Complex Does Not Require Preformed Actin Filaments (A) Time course of polymerization of 3 μM 15% pyrene actin with 50 nM S. pombe Arp2/3 complex and 200 nM Wsp1-VCA or the indicated concentrations of Dip1. Arrow highlights lag in activation of Arp2/3 complex by Wsp1-VCA. (B) Plot of branch density versus time for TIRF data in (C). Data are represented as mean ± SEM. (C) Total internal reflection microscopy (TIRF) images of 33% Oregon green 488 actin polymerizing with 50 nM S. pombe Arp2/3 complex, 150 nM Dip1, and 75 nM GST-Wsp1-VCA as indicated. The scale bar represents 2.2 μm. (D) Plot of total polymer length verses time for TIRF data in (C). (E) Plot of filament lengths versus time for select single filaments in TIRF data in (B). Dashed lines are linear fits of each filament growth. Global analysis of at least seven filaments/reaction showed that the average growth rate in reactions with Arp2/3 alone was 9.0 ± 0.1 s−1 (n = 541), Dip1 alone was 9.7 ± 0.3 s−1 (n = 816), Arp2/3 + GST-Wsp1-VCA was 9.2 ± 0.2 s−1 (n = 641), and Arp2/3 + dip1 was 9.5 ± 0.2 s−1 (n = 775). See also Movies S1 and S2. Current Biology 2013 23, 1990-1998DOI: (10.1016/j.cub.2013.08.029) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 Bulk Polymerization Assays Verify Preformed Filaments Are Not Required for Dip1-Mediated Arp2/3 Complex Activation (A) Pyrene actin polymerization assay showing the influence of 1.5 μM Crn1 WD-CC construct (contains residues 1–410 and 594–651) on activation of 50 nM S. pombe Arp2/3 complex by 5 μM Dip1 or 200 nM GST-Wsp1-VCA. (B) Pyrene actin polymerization assay showing the influence of preformed actin filaments on Dip1- versus Wsp1-activated Arp2/3 complex. Reactions contained 50 nM S. pombe Arp2/3 complex, 1 μM Dip1, 200nM GST-Wsp1-VCA, and 300 nM actin filament seeds as indicated. Current Biology 2013 23, 1990-1998DOI: (10.1016/j.cub.2013.08.029) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 Dip1 Uses a Non-WASP-like Mechanism to Activate Arp2/3 Complex (A) Western blot of supernatant and pelleted fractions in actin monomer pull-down assay. Actin at 1.0 μM was pulled down with 10 μM GST-Wsp1-VCA, 10 or 13.5 μM GST-Dip1, or 10 μM GST control protein. Quantified data are represented as the mean ± SEM (n = 3); asterisks indicate significant difference compared to GST control p < 0.0001 (parametric two-tailed t test). (B) Coomassie-stained native gel shift binding assay. Reactions contained indicated concentrations of each protein plus 40 μM Latrunculin B to prevent actin polymerization. (C) Coomassie-stained SDS-PAGE gel of actin filament copelleting assay. Dip1 (750 nM) or cortactin (750 nM) were copelleted with a range of concentrations of polymerized actin (total actin concentration is indicated). (D) Anti-Arp3 western blot of pull-down assay containing GST-Dip1 and 1.14 μM S. pombe Arp2/3 complex. Control assays contained 11 μM GST or 11 μM GST-Wsp1-VCA. Quantified data are represented as the mean ± SEM (n = 3); asterisks indicate significant difference compared to GST control p < 0.05 (parametric two-tailed t test). (E and F) Time courses of polymerization of 3 μM 15% pyrene actin with 50 nM S. pombe Arp2/3 complex, 7.5 μM profilin, and mutant or wild-type Dip1, as indicated. The concentration of Dip1 in (F) is 5 μM. See also Figure S2. Current Biology 2013 23, 1990-1998DOI: (10.1016/j.cub.2013.08.029) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 Dip1 Stimulates Formation of the Short Pitch Conformation (A) Cartoon schematic of Arp2/3 complex in the splayed or short pitch conformation showing relative positions of engineered cysteine residues (marked by X). (B) Anti-Arp3 western blot of crosslinking assays containing 1.0 μM S. cerevisiae Arp2/3 complex (Arp3L155C/Arp2R198C) and 25 μM BMOE, 10 μM leucine zipper (LZ) N-WASP-VCA, 10 μM latrunculin B-bound actin, and Dip1 as indicated. Reactions were allowed to proceed for 60 s before being quenched with 1.25 mM dithiolthreitol and separated by SDS-PAGE. (C) Quantification of short-pitch Arp2-Arp3 crosslinking assays as described in (B). Data are represented as mean ± SEM. p value was calculated from a parametric two-tailed t test. See also Figure S3. Current Biology 2013 23, 1990-1998DOI: (10.1016/j.cub.2013.08.029) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 6 SPIN90 and Dip1 May Use the Same Mechanism to Activate Arp2/3 Complex (A) Time course of 3 μM 15% pyrene actin polymerization showing influence of 2.2 μM wild-type or truncated GST-Dip1 on activation of 50 nM S. pombe Arp2/3 complex. (B) Time course of 3 μM 15% pyrene actin polymerization containing SPIN90 (residues 269–722) or GST-N-WASP-VCA and 50 nM B. taurus Arp2/3 (BtArp2/3) complex. (C) Pyrene actin polymerization assays containing 50 nM BtArp2/3 complex, 2 μM Crn1 WD-CC construct, 17.8 μM SPIN90, and 200 nM GST-N-WASP-VCA as indicated. (D) Pyrene actin polymerization assay showing the influence of preformed actin filaments on SPIN90 versus GST-N-WASP-VCA activated bovine Arp2/3 complex. Reactions contained 50 nM bovine Arp2/3 complex, 10.6 μM SPIN90, 200 nM GST-N-WASP-VCA, and 300 nM actin filament seeds as indicated. (E) Total internal reflection microscopy (TIRF) images of 33% Oregon green 488 actin polymerizing with BtArp2/3 complex and 1.5 μM SPIN90 or 100 nM GST-N-WASP-VCA as indicated. The reaction with 1.5 μM SPIN90 contains 25 nM BtArp2/3, and the reaction with N-WASP-VCA contains 20 nM BtArp2/3 complex. The scale bar represents 2.2 μm. (F) Plot of maximum polymerization rate versus SPIN90 concentration for the pyrene actin polymerization assays described in (B). See also Figure S4 and Movies S3 and S4. Current Biology 2013 23, 1990-1998DOI: (10.1016/j.cub.2013.08.029) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 7 Cartoon Model of Initiation and Propagation of Arp2/3-Mediated Branching Nucleation by Dip1 and Wsp1 See the main text for details. Current Biology 2013 23, 1990-1998DOI: (10.1016/j.cub.2013.08.029) Copyright © 2013 Elsevier Ltd Terms and Conditions